1. Field of the Invention
This invention relates to an improved assay method and reagent composition for determining creatinine levels in body fluids, and more particularly to a colorimetric method which is performed directly on serum and other fluids without deproteinization.
2. Nature and Significance of Accurate Creatinine Determinations
It has been recognized for almost a century that the measurement of creatinine is of vital medical significance, and even today, the most widely used kidney function test in clinical practice is the estimation of glomerular filtration rate by measurement of the creatinine level in serum and urine specimens. Tests or assays that measure such creatinine levels are major criteria in determining whether kidney disease is present and, if present, the extent to which it is present.
Creatinine is synthesized in the body at a fairly constant rate from creatine, which is produced during muscle contractions from creatine phosphate. Creatinine in the blood is then removed by filtration through the glomeruli of the kidney for excretion in the urine. Since the excretion of creatinine in healthy individuals is independent of diet and thus relatively constant, the creatinine clearance test is one of the most sensitive tests for measuring glomerular filtration rate, and the concentration of creatinine in the serum depends almost entirely upon its rate of excretion by the kidney. In kidney disease, therefore, creatinine levels in the blood are elevated, while the creatinine clearance, or urine levels, are diminished.
3. Discussion of the Prior Art
Prior art methods date as far back as 1886 when Jaffe (Z. Physiol. Chem. 10:391, 1886) described the formation of a red color when creatinine was reacted with picrate in an alkaline solution. Today, this classic Jaffe reaction remains the basis for a majority of the current creatinine assay methods.
Although the Jaffe reaction achieves a measurable result, it has long been recognized that the reaction suffers a significant drawback in its lack of specificity for measuring creatinine in serum and urine. More particularly, a number of other substances which are normally present in serum and urine also form a red color with picrate or otherwise interfere with the reaction, thereby causing inaccurate and misleading results. Most of these substances cause a positive interference by increasing the amount of color formation. Such substances include, for example, protein, glucose, ascorbate, acetoacetate, pyruvate and other alpha-keto acids. On the other hand, bilirubin in the presence of protein causes a negative interference, or falsely low results.
To overcome the problems of non-specific color formation, numerous methods have been developed which remove the interfering substance by some type of sample pretreatment, the most commonly performed pretreatment methods being protein precipitation and removal by filtration, treatment with Lloyd's reagent, or removal of protein by dialysis through a membrane.
However, such pretreatment methods are cumbersome and time-consuming. Furthermore, with the exception of protein removal by dialysis, pretreatment methods have a particular disadvantage in that they do not easily lend themselves to automation, which has become a virtual necessity in meeting the demands of today's clinical laboratories. Additionally, pretreatment in many cases may solve only a portion of the nonspecificity problems or may even create additional specificity problems.
In 1971, Bartels and Bohmer (Clin. Chem. Acta 32:81, 1971) and Cook (Clin. Chim. Acta 32:485, 1971) proposed the measurement of creatinine in the presence of protein by measuring the rate of color development of the classic Jaffe reaction, i.e., kinetic measurement. Although many different means have been attempted in past years to improve the specificity of the Jaffe reaction, the most popular means of improving specificity of creatinine assays has been the use of kinetic methods based on the Jaffe reaction. Denney and Long (U.S. Pat. No. 4,111,657, issued Sept. 5, 1978) suppressed protein interference in a kinetic creatinine assay by the incorporation of dimethylsulfoxide in an alkaline picrate reagent. Although their method achieved several important advantages over earlier attempts, their modifications left unsolved the problem of the negative interference from bilirubin present in serum specimens, i.e. icteric samples. Many investigators have recently studied and attempted to correct for this negative interference, which appears to exist in virtually all of today's popularly used creatinine assay methods which are carried out in the presence of protein.
Two investigators, Tagesson and Rebel (Clin. Chem. 26:520, 1980), quite apart from attempting to solve bilirubin interference, attempted to resolve a problem of drift (a continuous gradual increase in results) in certain continuous flow creatinine methods. (Continuous flow methods rely upon the separation of protein from the specimens by use of a semi-permeable membrane.) Tagesson and Rebel postulated that calcium was precipitating in the cuvette in which optical density was measured and added EDTA to prevent the precipitation of calcium and thus reduce the drift causing increasing creatinine values. The present invention also incorporates EDTA, however achieving benefits quite in contrast to those of Tagesson and Rebel. In fact, the benefits of the present invention would be quite unnoticed and unappreciated in continuous flow methods since protein, and substances which are bound to protein, including bilirubin, are separated and removed from the samples being assayed.